Significance of Antibody Orientation Unraveled: Well-Oriented Antibodies Recorded High Binding Affinity

To investigate the effect of antibody. orientation on its immunological activities, we developed a novel and versatile platform consisting of a well-defined phospholipid polymer surface on which staphylococcal protein A (SpA) was site-selectively immobilized. The application of a biocompatible phospholipid-based platform ensured minimal denaturation of immobilized antibodies, and the site selective immobilization of SpA clarified the effect of antibody orientation on immunological activities. The phospholipid polymer platform Was prepared on silicon substrates using the surface initiated atom transfer radical polymerization (SI-ATRP) technique. An enzymatic reaction was performed for orientation-selective coupling of SpA molecules to the polymer brush surface. Orientation-controlled antibodies were achieved using enzymatic reactions, and these antibodies captured 1.8 +/- 0.1 antigens on average, implying that at least 80% of immobilized antibodies reacted with two antigens. Theoretical multivalent binding analysis further revealed that orientation-controlled antibodies had antigen-antibody reaction equilibrium dissociation constants (K-d) as low as 8.6 x 10(-10) mol/L, whereas randomly oriented and partially oriented antibodies showed K-d values of 2.0 x 10(-7) and 1.2 x 10(-7) mol/L, respectively. Strict control of antibody orientation not only formed an approximately 100-fold stronger antigen-antibody complex than the controls but also sustained the native antibody K-d (10(-10)-10(-9) mol/L). These findings support the significance of antibody orientation because controlling the orientation resulted in high reactivity and theoretical binding capacity.